Method for the purification of albumin conjugates

ABSTRACT

The present invention relates to a method for separating albumin conjugate from unconjugated albumin in a solution comprising albumin conjugate and unconjugated albumin by loading the solution onto a hydrophobic support equilibrated in aqueous buffer having a high salt content; applying to the support a gradient of decreasing salt concentration; and collecting the eluted albumin conjugate.

RELATED APPLICATION

This application claims priority to U.S. provisional patent applicationSer. No. 60/565,228 filed Apr. 23, 2004, which is expressly incorporatedby reference in its entirety.

BACKGROUND OF THE INVENTION

(a) Field of the Invention

This invention relates to a method of purification for isolating albuminconjugates from a solution comprising both albumin conjugates andunconjugated albumin.

(b) Description of Prior Art

WO 95/10302 and WO 99/24074 describe the formation of conjugates ofalbumin wherein the molecule of interest has a reactive functionalitycoupled thereto that is adapted to covalently bond to albumin, thusforming a conjugate. These conjugates can be formed in vivo, but theycan be formed in vitro as well. The formation of the conjugate in vitroinvolves the addition of a molecule coupled to a reactive functionalityto a solution of albumin. The primary end products from this reactionare unconjugated albumin, the albumin conjugate and the unreactedmolecule coupled to the reactive functionality.

It would be highly desirable to be provided with a method for purifyingalbumin conjugate from a solution comprising albumin conjugate andunconjugated albumin.

SUMMARY OF THE INVENTION

In accordance with the present invention there is provided a method forseparating albumin conjugate from unconjugated albumin in a solutioncomprising albumin conjugate and unconjugated albumin, the methodcomprising:

a) loading the solution onto a hydrophobic solid support equilibrated inaqueous buffer having a high salt content;b) applying to the support a gradient of decreasing salt content; andc) collecting eluted albumin conjugate.

In a preferred embodiment of the present invention, the albuminconjugate consists of a molecule having a Michael acceptor covalentlycoupled thereto which bonds to albumin, and more preferably the bond isbetween the Michael acceptor and cysteine 34 of albumin.

In a more preferred embodiment of the present invention, the Michaelacceptor is a maleimide group, and more preferably, the maleimide groupis maleimide-propionic acid (MPA). The Michael acceptor is optionallycoupled to the molecule via a linker. The linker is preferably selectedin the group consisting of hydroxyethyl motifs such as (2-amino) ethoxyacetic acid (AEA), ethylenediamine (EDA), 2-[2-(2-amino)ethoxy)]ethoxyacetic acid (AEEA), amino ethoxy ethyl amino succinic acid (AEEAS); oneor more alkyl chains (C1-C10) motifs such as glycine, 3-aminopropionicacid (APA), 8-aminooctanoic acid (AOA), octanoic acid (OA),4-aminobenzoic acid (APhA). Preferred linkers are OA, ADE, AEA, AEEA andAEEAS. A combination of two linkers can also be used such as, forexamples, AEEA-EDA, AEEA-AEEA, AEEAS-AEEAS, and AEA-AEEA.

In a preferred embodiment of the present invention, the albumin isselected from the group consisting of serum albumin, recombinant albuminand albumin from a genomic source.

In a preferred embodiment of the present invention, the albumin isselected from the group consisting of human albumin, rat albumin, mousealbumin, swine albumin, bovine albumin, dog albumin and rabbit albumin,more preferable human serum albumin.

In a preferred embodiment, albumin is modified with at least oneselected from the group consisting of fatty acids, metal ions, smallmolecules having high affinity to albumin, and sugars, such as, but notlimited to, glucose, lactose and mannose.

In a preferred embodiment of the present invention, the molecule isselected from the group consisting of a peptide, DNA, RNA, small organicmolecule and a combination thereof. The peptide has preferentially amolecular weight of at least 57 daltons. The peptide is intended toinclude, but not being limited to, GLP-1, GLP-2, ANP, K5, dynorphin,GRF, insulin, natriuretic peptides, T-20, T-1249, C-34 and PYY. Thesmall molecule is intended to include, but not being limited to,vinorelbine, gemcitabine and paclitaxel. In a more preferred embodimentof the present invention, when the molecule is a DNA, RNA or a smallorganic molecule, it is covalently attached to the albumin through anacid sensitive covalent bond or a peptide sequence susceptible toproteolytic cleavage, thereby allowing the separation of the moleculefrom albumin and the entry of the molecule into a cell.

In a preferred embodiment of the present invention, the hydrophobicsolid support is a column containing a hydrophobic resin such as, butnot limited to, octyl sepharose, phenyl sepharose and butyl sepharoseand more preferably butyl sepharose.

In another embodiment of the present invention, the hydrophobic solidsupport comprising a hydrophobic ligand such as Cibacron Blue F3G-A,ether or isopropyl groups in association with a support such aspolystyrene/divinyl benzene matrix.

Substances are separated on the basis of their varying strengths ofhydrophobic interactions with hydrophobic ligands immobilized to anuncharged matrix. This technique is usually performed with moderatelyhigh concentrations of salts (≈1M) in the start buffer (salt promotedadsorption). Elution is achieved by a linear or stepwise decrease insalt concentration.

The type of ligand, the degree of substitution, the pH and the type andconcentration of salt used during the adsorption stage have a profoundeffect on the overall performance (e.g. selectivity and capacity) of aHIC matrix (Hydrophobic Interaction Chromatography matrix).

The solvent is one of the most important parameters which influencecapacity and selectivity in HIC (Hydrophobic InteractionChromatography). In general, the adsorption process is more selectivethan the desorption process. It is therefore important to optimize thestart buffer with respect to pH, type of solvent, type of salt andconcentration of salt. The addition of various “salting-out” salts tothe sample promotes ligand-protein interactions in HIC. As theconcentration of salt is increased, the amount of bound proteinincreases up to the precipitation point for the protein. Each type ofsalt differs in its ability to promote hydrophobic interactions. Theinfluence of different salts on hydrophobic interaction follows thewell-known Hofmeisters series found below:

Hofmeisters Series Salting-Out Effect Anions:

PO₄ ³⁻>SO₄ ²⁻>CH₃COO⁻>Cl⁻>Br⁻>NO₃ ⁻>ClO₄ ⁻>I⁻>SCN⁻

Chaotropic Effect Cations: NH₄ ⁺<Rb⁺<K⁺<Na⁺<Cs⁺<Li⁺<Mg²⁺<Ba²⁺

Increasing the salting-out effect strengthens the hydrophobicinteractions, whereas increasing the chaotropic effect weakens them.Therefore, ammonium sulfate exhibits a stronger salting-out effect thansodium chloride. The most commonly used salts for HIC are ammoniumsulfate ((NH₄)₂SO₄), sodium sulfate ((Na)₂SO₄)), magnesium sulfate(MgSO₄), sodium chloride (NaCl), potassium chloride (KCl), and ammoniumacetate (CH₃COONH₄).

Protein binding to HIC adsorbents is promoted by moderate to highconcentrations of “salting-out” salts, most of which also have astabilizing influence on protein structure due to their preferentialexclusion from native globular proteins, i.e. the interaction betweenthe salt and the protein surface is thermodynamically unfavorable. Thesalt concentration should be high enough (e.g. 500-1000 mM) to promoteligand-protein interactions yet below that which causes precipitation ofthe protein in the sample. In the case of albumin, the saltconcentration should be kept below 3M (moles per liter). The principlemechanism of salting-out consists of the salt-induced increase of thesurface tension of water (Melander and Horváth, 1977). Thus, a compactstructure becomes energetically more favorable because it corresponds tosmaller protein-solution interfacial area.

Interestingly, we found that under the same conditions (i.e. buffercomposed of SO₄ ²⁻, PO₄ ²⁻ or CH₃COO⁻ with any counter ion), these saltsexhibit their salting-out effect upon essentially all conjugated albumindescribed herein in a manner different to non-conjugated albumin (i.e.mercaptalbumin and albumin capped with cysteine), thus enabling aconsistent chromatographic separation between conjugated albumin versusnon-conjugated albumin. That is, we observe that lower concentrations ofsalt are required to promote interactions between ligand and conjugatedalbumin than between ligand and non-conjugated albumin. Thischromatographic separation is essentially independent of (a) thesequence of albumin (e.g. human, mouse, rat, etc.) (b) the source ofalbumin (i.e. plasma derived or recombinant) (c) the molecular weight ofthe conjugated molecule, (d) the position of the Michael acceptor (ormaleimide group) within the structure of the molecule, (e) the peptidesequence or chemical structure of the molecule, and (f) thethree-dimensional structure of the conjugated molecule, e.g. linearversus loop structure.

In a preferred embodiment of the present invention, the salt of theaqueous buffer has a sufficient salting out effect. For providing asufficient salting out effect, the salt is preferably, but not limitedto, phosphate, sulfate and acetate. More preferably, the salt isphosphate or sulfate. The selection of the cation of the buffer is lesscritical and therefore, such cation can be selected, without limitation,from the group consisting of NH₄ ⁺, Rb⁺, K⁺, Na⁺, Cs⁺, Li⁺, Mg²⁺ andBa²⁺.

The aqueous buffer is preferably ammonium phosphate, ammonium sulfateand magnesium phosphate, and more preferably ammonium sulfate.

In a preferred embodiment of the present invention, the buffer pH isbetween 3.0 and 9.0; more preferably between 6.0 and 8.0, and even morepreferably, the pH is 7.0.

In a preferred embodiment of the present invention, the buffer and thehydrophobic solid support are at room temperature (about 25° C.) or at4° C. or in between.

Table 1 shows an example of the effect of varying salts for purificationof preformed HSA:first GLP-1 analogue conjugate from a solution of HSAusing butyl-sepharose resin (structure of the first GLP-1 analogue isdescribed in Example 1 below).

TABLE 1 Starting salt Starting salt concentration of concentration ofSalt type 750 mM 1,750 mM Ammonium phosphate Yes yes Ammonium sulfateYes yes Ammonium chloride No no Ammonium iodide No no Ammoniumthiocyanate No no Magnesium sulfate No yes Magnesium phosphate* — —Barium sulfate* — — *means that the salt is not soluble atconcentrations of 1750 mM or 750 mM in 20 mM sodium phosphate (pH 7), 5mM caprylate Yes means that successful resolution is achieved betweenthe HSA:first GLP-1 analogue conjugate and the non-conjugated HSA Nomeans that no separation is achieved between the HSA:first GLP-1analogue conjugate and the non-conjugated HSA

The term “peptide” is intended to mean an amino acid sequence having amolecular weight of at least 57 daltons. The peptidic sequence can becircular (loop structure) such as ANP, may contain more than one aminoacid chain such as insulin or may be linear such as K5, dynorphin A,C-34 and GLP-1.

All references herein are hereby incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the purification of the conjugate HSA:first GLP-1analogue (SEQ ID NO:1) by a preferred embodiment of the method of thepresent invention;

FIG. 2 illustrates the purification of the conjugate HSA:first GRFanalogue (SEQ ID NO:2) by a preferred embodiment of the method of thepresent invention;

FIG. 3 illustrates the purification of non-conjugated HSA by a preferredembodiment of the method of the present invention;

FIG. 4 illustrates the purification of the conjugate rHSA:first GLP-1analogue (SEQ ID NO:1) by a preferred embodiment of the method of thepresent invention;

FIG. 5 illustrates the purification of HSA cortex by a preferredembodiment of the method of the present invention;

FIG. 6 illustrates the purification of the conjugate HSA:K5 analogue(SEQ ID NO:3) by a preferred embodiment of the method of the presentinvention;

FIG. 7 illustrates the purification of the conjugate HSA:first insulinderivative (SEQ ID NO:4) by a preferred embodiment of the method of thepresent invention;

FIG. 8 illustrates the purification of the conjugate HSA:second insulinderivative (SEQ ID NO:5) by a preferred embodiment of the method of thepresent invention;

FIG. 9 illustrates the purification of the conjugate HSA:first C34analogue (SEQ ID NO:6) by a preferred embodiment of the method of thepresent invention;

FIG. 10 illustrates the purification of the conjugate HSA:second C34analogue (SEQ ID NO:7) by a preferred embodiment of the method of thepresent invention;

FIG. 11 illustrates the purification of the conjugate HSA:third C34analogue (SEQ ID NO:8) by a preferred embodiment of the method of thepresent invention;

FIG. 12 illustrates the purification of L-cysteine by a preferredembodiment of the method of the present invention;

FIG. 13 illustrates the purification of L-cysteine:first GLP-1 analogue(SEQ ID NO:1) by a preferred embodiment of the method of the presentinvention;

FIG. 14 illustrates the purification of the conjugate HSA:second GLP-1analogue (SEQ ID NO:9) by a preferred embodiment of the method of thepresent invention;

FIG. 15 illustrates the purification of the conjugate HSA:third GLP-1analogue (SEQ ID NO:10) by a preferred embodiment of the method of thepresent invention;

FIG. 16 illustrates the purification of the conjugate HSA:fourth GLP-1analogue (SEQ ID NO:11) by a preferred embodiment of the method of thepresent invention;

FIG. 17 illustrates the purification of the conjugate HSA:fifth GLP-1analogue (SEQ ID NO:12) by a preferred embodiment of the method of thepresent invention;

FIG. 18 illustrates the purification of the conjugate HSA:firstExendin-4 analogue (SEQ ID NO:13) by a preferred embodiment of themethod of the present invention;

FIG. 19 illustrates the purification of the conjugate HSA:secondExendin-4 analogue (SEQ ID NO:14) by a preferred embodiment of themethod of the present invention;

FIG. 20 illustrates the purification of HSA:MPA by a preferredembodiment of the method of the present invention;

FIG. 21 illustrates the purification of HSA by a preferred embodiment ofthe method of the present invention;

FIG. 22 illustrates the purification of the conjugate HSA:second C34analogue (SEQ ID NO:3) by a preferred embodiment of the method of thepresent invention;

FIG. 23 illustrates the purification of the conjugate HSA:firstDynorphin A analogue (SEQ ID NO:15) by a preferred embodiment of themethod of the present invention;

FIG. 24 illustrates the purification of the conjugate HSA:first ANPanalogue (SEQ ID NO:16) by a preferred embodiment of the method of thepresent invention;

FIG. 25 illustrates the purification of the conjugate HSA:secondDynorphin A analogue (SEQ ID NO:17) by a preferred embodiment of themethod of the present invention;

FIG. 26 illustrates the purification of the conjugate HSA:ACE inhibitor(SEQ ID NO:18) by a preferred embodiment of the method of the presentinvention;

FIG. 27 illustrates the purification of the conjugate HSA:sixth GLP-1analogue (SEQ ID NO:19) by a preferred embodiment of the method of thepresent invention;

FIG. 28 illustrates the purification of the conjugate HSA:seventh GLP-1analogue (SEQ ID NO:20) by a preferred embodiment of the method of thepresent invention;

FIG. 29 illustrates the purification of the conjugate HSA:eighth GLP-1analogue (SEQ ID NO:21) by a preferred embodiment of the method of thepresent invention;

FIG. 30 illustrates the purification of the conjugate HSA:ninth GLP-1analogue (SEQ ID NO:22) by a preferred embodiment of the method of thepresent invention;

FIG. 31 illustrates the purification of the conjugate HSA:tenth GLP-1analogue (SEQ ID NO:23) by a preferred embodiment of the method of thepresent invention;

FIG. 32 illustrates the purification of the conjugate HSA:eleventh GLP-1analogue (SEQ ID NO:24) by a preferred embodiment of the method of thepresent invention;

FIG. 33 illustrates the purification of the conjugate HSA:thirdExendin-4 analogue (SEQ ID NO:25) by a preferred embodiment of themethod of the present invention;

FIG. 34 illustrates the purification of the conjugate HSA:twelfth GLP-1analogue (SEQ ID NO:26) by a preferred embodiment of the method of thepresent invention;

FIG. 35 illustrates the purification of the conjugate HSA:first insulinderivative (SEQ ID NO:4) by a preferred embodiment of the method of thepresent invention;

FIG. 36 illustrates the purification of the conjugate HSA:third insulinderivative (SEQ ID NO:27) by a preferred embodiment of the method of thepresent invention;

FIG. 37 illustrates the purification of the conjugate HSA:second insulinderivative (SEQ ID NO:5) by a preferred embodiment of the method of thepresent invention;

FIG. 38 illustrates the purification of the conjugate HSA:fourth insulinderivative (SEQ ID NO:28) by a preferred embodiment of the method of thepresent invention;

FIG. 39 illustrates the purification of the conjugate HSA:first GRFanalogue (SEQ ID NO:2) by a preferred embodiment of the method of thepresent invention;

FIG. 40 illustrates the purification of the conjugate HSA:second GRFanalogue (SEQ ID NO:29) by a preferred embodiment of the method of thepresent invention;

FIG. 41 illustrates the purification of the conjugate HSA:third GRFanalogue (SEQ ID NO:30) by a preferred embodiment of the method of thepresent invention;

FIG. 42 illustrates the purification of the conjugate HSA:fourth GRFanalogue (SEQ ID NO:31) by a preferred embodiment of the method of thepresent invention;

FIG. 43 illustrates the purification of the conjugate HSA:thirteenthGLP-1 analogue CJC 1365 (SEQ ID NO:32) by a preferred embodiment of themethod of the present invention;

FIG. 44 illustrates the purification of the conjugate HSA lactose:firstGLP-1 analogue (SEQ ID NO:1) by a preferred embodiment of the method ofthe present invention;

FIG. 45 illustrates the purification of the conjugate HSA:first T20analogue (SEQ ID NO:33) by a preferred embodiment of the method of thepresent invention;

FIG. 46 illustrates the purification of the conjugate HSA:first T1249analogue (SEQ ID NO:34) by a preferred embodiment of the method of thepresent invention;

FIG. 47 illustrates the purification of the compound HSA:first GLP-1analogue (SEQ ID NO:1) by a preferred embodiment of the method of thepresent invention;

FIG. 48 illustrates the purification of the compound HSA:first C34analogue (SEQ ID NO:6) by a preferred embodiment of the method of thepresent invention;

FIG. 49 illustrates the purification of the compound HSA:second GRFanalogue (SEQ ID NO:29) by a preferred embodiment of the method of thepresent invention;

FIG. 50 illustrates the purification of the conjugate HSA:vinorelbineanalogue conjugate (SEQ ID NO:35) by a preferred embodiment of themethod of the present invention;

FIG. 51 illustrates the purification of L-cysteine by a preferredembodiment of the method of the present invention;

FIG. 52 illustrates the purification of the conjugateL-Cysteine:vinorelbine analogue (SEQ ID NO:35) by a preferred embodimentof the method of the present invention;

FIG. 53 illustrates the purification of the conjugate RSA:thirdExendin-4 analogue (SEQ ID NO:25) by a preferred embodiment of themethod of the present invention;

FIG. 54 illustrates the purification of the conjugate HSA:fourth C34analogue (SEQ ID NO:36) by a preferred embodiment of the method of thepresent invention;

FIG. 55 illustrates the purification of the conjugate HSA:fifth C34analogue (SEQ ID NO:37) by a preferred embodiment of the method of thepresent invention;

FIG. 56 illustrates the purification of the conjugate HSA:sixth C34analogue (SEQ ID NO:38) by a preferred embodiment of the method of thepresent invention;

FIG. 57 illustrates the purification of the conjugate HSA:seventh C34analogue (SEQ ID NO:39) by a preferred embodiment of the method of thepresent invention;

FIG. 58 illustrates the purification of the conjugate HSA:eighth C34analogue (SEQ ID NO:40) by a preferred embodiment of the method of thepresent invention;

FIG. 59 illustrates the purification of the conjugate HSA:first PYYanalogue (SEQ ID NO:41) by a preferred embodiment of the method of thepresent invention;

FIG. 60 illustrates the purification of the conjugate HSA:second PYYanalogue (SEQ ID NO:42) by a preferred embodiment of the method of thepresent invention;

FIG. 61 illustrates the purification of the conjugate HSA:fifth insulinderivative (SEQ ID NO:43) by a preferred embodiment of the method of thepresent invention;

FIG. 62 illustrates the purification of the conjugate HSA:sixth insulinderivative (SEQ ID NO:44) by a preferred embodiment of the method of thepresent invention;

FIG. 63 illustrates the purification of the conjugate HSA:seventhinsulin derivative (SEQ ID NO:45) by a preferred embodiment of themethod of the present invention;

FIG. 64 illustrates the purification of the conjugate HSA:third PYYanalogue (SEQ ID NO:46) by a preferred embodiment of the method of thepresent invention;

FIG. 65 illustrates the purification of the conjugate HSA:fourth PYYanalogue (SEQ ID NO:47) by a preferred embodiment of the method of thepresent invention;

FIG. 66 illustrates the purification of the conjugate HSA:fifth PYYanalogue (SEQ ID NO:48) by a preferred embodiment of the method of thepresent invention;

FIG. 67 illustrates the purification of the conjugate HSA:sixth PYYanalogue (SEQ ID NO:49) by a preferred embodiment of the method of thepresent invention;

FIG. 68 illustrates the purification of the conjugate HSA:second ANPanalogue (SEQ ID NO:50) by a preferred embodiment of the method of thepresent invention;

FIGS. 69A-B illustrates the purification of the conjugate HSA:third ANPanalogue CJC 1681 (SEQ ID NO:51) by a preferred embodiment of the methodof the present invention;

FIG. 70 illustrates the purification of the conjugate HSA:first GLP-1analogue (SEQ ID NO:1) by a preferred embodiment of the method of thepresent invention;

FIG. 71 illustrates the purification of the conjugate HSA:first GLP-1analogue (SEQ ID NO:1) by a preferred embodiment of the method of thepresent invention;

FIG. 72 illustrates the purification of the conjugate HSA:first GLP-1analogue (SEQ ID NO:1) by a preferred embodiment of the method of thepresent invention;

FIG. 73 illustrates the purification of the conjugate HSA:first GLP-1analogue (SEQ ID NO:1) by a preferred embodiment of the method of thepresent invention;

FIG. 74 illustrates the purification of the conjugate HSA:first GLP-1analogue (SEQ ID NO:1) by a preferred embodiment of the method of thepresent invention;

FIG. 75 illustrates the purification of the conjugate HSA:first GLP-2analogue (SEQ ID NO:52) by a preferred embodiment of the method of thepresent invention; and

FIG. 76 illustrates the purification of the conjugate RSA:first GLP-2analogue (SEQ ID NO:52) by a preferred embodiment of the method of thepresent invention.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT OF THE INVENTION

In accordance with the present invention, there is provided a method forpurifying albumin conjugates from a solution comprising albuminconjugates and unconjugated albumin.

Methods Preparation of Control (Non-Conjugated) Human Serum Albumin(HSA) and Preformed Albumin Conjugates

Each compound with the Michael acceptor was solubilized in nanopurewater (or in DMSO if the compound was difficult to solubilize) at aconcentration of 10 mM, then diluted to 1 mM into a solution of HSA(25%, 250 mg/ml, Cortex-Biochem, San Leandro, Calif.). The samples werethen incubated at 37° C. for 30 min. Prior to their purification, eachconjugate solution was diluted to 5% 50 mg/ml HSA in 20 mM sodiumphosphate buffer (pH 7) composed of 5 mM sodium octanoate. The initialconcentration of salt used in the elution gradient can be added to thebuffer for diluting the mixed solution. Preferably, the initialconcentration of salt is from about 750 to about 1700 mM (NH₄)₂SO₄.

Procedure for Purification According to a Preferred Embodiment

Using an

KTA purifier (Amersham Biosciences, Uppsala, Sweden), each conjugate wasloaded at a flow rate of 2.5 ml/min onto a 50 ml column of butylsepharose 4 fast flow resin (Amershan Biosciences, Uppsala, Sweden)equilibrated in 20 mM sodium phosphate buffer (pH 7) composed of 5 mMsodium octanoate and 750 mM to 1.7 M (NH₄)₂SO₄. Under these conditions,HSA conjugates having a molecular weight addition of more than 2 kDarelative to non-conjugated HSA adsorbed onto the hydrophobic resinwhereas essentially all non-conjugated HSA eluted within the void volumeof the column. For molecular weight additions of less than 2 kDa, ahigher initial salt content may be used followed by a stepwise gradientof decreasing salt. Each conjugate was further purified from any freeunconjugated compound by applying a continuous or non-continuousdecreasing gradient of salt (750 to 0 mM (NH₄)₂SO₄) over 4 columnvolumes. In a preferred embodiment, each purified conjugate was thendesalted and concentrated by diafiltration, for instance by usingAmicon® ultra centrifugal (30 kDa) filter devices (MilliporeCorporation, Bedford, Mass.). Finally, for prolonged storage, eachconjugate solution is preferably immersed into liquid nitrogen, andlyophilized using a Labconco freeze dry system (FreeZone®4.5), andstored at −20° C.

Examples of LC/EMS Analysis

Following purification, 1 μl of each conjugate sample is preferablyinjected onto LC/EMS system. The HSA:first GLP-1 analogue (SEQ ID NO:1)conjugate was confirmed by detection of a species of highest abundancewith a total mass of 70 160 Da which corresponds to the mass ofmercaptalbumin (66 448 Da) where cysteine 34 is in the free thiol form,plus the mass of only one molecule of the first GLP-1 analogue (3 719.9Da). The structure of the first GLP-1 analogue (SEQ ID NO:1) isdescribed in Example 1 below. This is illustrated in Table 2.

TABLE 2 Molecular Absolute Relative Component Weight Abundance AbundanceA 70160.58 321970 100.00 B 65862.95 70008 21.74 C 64545.45 62888 19.53 D70320.04 41167 12.79 E 61287.67 16842 5.23 F 60623.81 16522 5.13 G58090.04 12473 3.87

The HSA:first GRF analogue (SEQ ID NO:2) conjugate was confirmed bydetection of a species of highest abundance with a total mass of 70 086Da which corresponds to the mass of mercaptalbumin (66 448 Da) wherecysteine 34 is in the free thiol form, plus the mass of only onemolecule of the first GRF analogue (3648.2 Da). The structure of thefirst GRF analogue (SEQ ID NO:2) is described in Example 2 below. Thisis illustrated in Table 3.

TABLE 3 Molecular Absolute Relative Component Weight Abundance AbundanceA 70086.06 279413 100.00 B 63214.84 53333 19.09 C 62148.17 38582 13.81 D70247.98 34870 12.48 E 56795.96 10523 3.77 F 62695.49 9813 3.51

The following examples illustrate several compounds having a maleimidegroup as Michael acceptor that have been conjugated to albumin andpurified in accordance with the method of the present invention.

The following examples are for the purpose of illustrating the presentinvention and not of limiting its scope.

In the following examples, the gradient numbers refer to the followinggradient details, where CV means a column volume of 50 ml.

Gradient #1: Linear 750-0 mM (NH₄)₂SO₄, over 4 CV, flow rate of 2.5ml/min.

Gradient #2: Step gradient 1.75M-1.2M (NH₄)₂SO₄ over 0.5 CV, followed by1.2M-875 mM (NH₄)₂SO₄ over 5 CV, and finally 875 mM-0 mM (NH₄)₂SO₄ over0.5 CV flow rate of 2.5 ml/min.

Gradient #3: Linear 900-0 mM (NH₄)₂SO₄ over 4 CV, flow rate of 2.5ml/min.

Gradient #4: Step gradient 1.5M-1.1M (NH₄)₂SO₄ over 0.5 CV, followed by1.1M-375 mM (NH₄)₂SO₄ over 6 CV, and finally 375 mM-0 mM (NH₄)₂SO₄ over0.5 CV, flow rate of 2.5 ml/min.

Gradient #5: Linear 750-0 mM (NH₄)₂SO₄ over 2 CV, flow rate of 2.5ml/min.

Gradient #6: Step gradient 1.75M-0M (NH₄)₂SO₄ over 6 CV, flow rate of2.5 ml/min.

Gradient #7: Linear 750-0 mM (NH₄)₂SO₄ over 6 CV, flow rate of 2.5ml/min.

Example 1 Purification of HSA:First GLP-1 Analogue (SEQ ID NO:1)Conjugate

The first GLP-1 analogue is GLP-1 (7-36) dAla⁸ Lys³⁷ (ε-AEEA-MPA)-CONH₂and has the following sequence:

H(dA)EGTFTSDVSSYLEGQAAKEFIAWLVKGRK(AEEA-MPA)-CONH₂

The purification of a conjugate made from reacting 1 ml 25% 250 mg/mlHSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM first GLP-1 analoguediluted into 9 ml of buffer made of 20 mM sodium phosphate buffer pH7.0, 5 mM sodium caprylate and 750 mM (NH₄)₂SO₄, was performed on acolumn of Butyl sepharose using the gradient #1 described above. In FIG.1 the purified conjugate fraction elutes during the gradient ofdecreasing (NH₄)₂SO₄ concentration as fraction B (F8-F9), whereasnon-conjugated albumin elutes within the void volume of the column(fraction A). The conjugate fraction was concentrated with Ultrafree™filter 30 kDa and analyzed using LC-EMS.

Example 2 Purification of HSA:First GRF Analogue (SEQ ID NO:2) Conjugate

The first GRF analogue is GRF (1-29) dAla² Gln⁸ Ala¹⁵ Leu²⁷ Lys³⁰(ε-MPA) CONH₂ and has the following sequence:

YaDAIFTQSYRKVLAQLSARKLLQDILSRK(MPA)-CONH₂

The purification of a conjugate made from reacting 1 ml 25% 250 mg/mlHSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM first GRF analoguediluted into 9 ml of 20 mM sodium phosphate buffer (pH 7.0), 5 mM sodiumcaprylate and 750 mM (NH₄)₂SO₄, was performed on a column of Butylsepharose using the gradient #1 described above. In FIG. 2 the purifiedconjugate fraction appears in fraction B (F6-F7) whereas non-conjugatedalbumin elutes within the void volume of the column (fraction A). Theconjugate fraction was concentrated with Ultrafree™ filter 30 kDa andanalyzed using LC-EMS.

Example 3 Purification of Non-Conjugated HSA 1 ml

The purification of 1 ml 25% 250 mg/ml non-conjugated HSA(Cortex-Biochem, San Leandro, Calif.) diluted into 9 ml of buffer (pH7.0) made of 20 mM sodium phosphate buffer, 5 mM sodium caprylate and750 mM (NH₄)₂SO₄, was performed on a column of Butyl sepharose using thegradient #1 described above. Essentially all albumin molecules elutewithin the void volume and no protein species is observed at 280 nmduring (NH₄)₂SO₄ gradient. FIG. 3 illustrates the separation curveobtained.

Example 4 Purification of rHSA:First GLP-1 Analogue (SEQ ID NO:1)Conjugate

The first GLP-1 analogue is GLP-1 (7-36) dAla⁸ Lys³⁷ (ε-AEEA-MPA)-CONH₂and his sequence is shown in Example 1.

The purification of a conjugate made from reacting 5 ml 5% rHSA(recombinant HSA new century culture grade) with 200 μM first GLP-1analogue diluted into 5 ml of a buffer made of 20 mM sodium phosphatebuffer, 5 mM sodium caprylate and 750 mM (NH₄)₂SO₄ was performed on acolumn of Butyl sepharose using the gradient #1 described above. In FIG.4 the purified conjugate fraction appears in fraction B (F7-F8-F9).

Example 5 Purification of HSA 10 ml

The purification of 10 ml 25% 250 mg/ml HSA (Cortex-Biochem, SanLeandro, Calif.) diluted into 40 ml of a buffer made of 20 mM sodiumphosphate buffer (pH 7.0), 5 mM sodium caprylate and 750 mM (NH₄)₂SO₄was performed on a column of Butyl sepharose using the gradient #1described above. Essentially all albumin molecules elute within a voidvolume and no protein species is observed at 280 nm during (NH₄)₂SO₄gradient. FIG. 5 illustrates the separation curve obtained.

Example 6 Purification of HSA:K5 Analogue (SEQ ID NO:3) Conjugate

The K5 analogue is Ac-K5 Lys⁸ (ε-MPA)-NH₂ and has the followingsequence:

The purification of a conjugate made from reacting 4 ml 25% 250 mg/mlHSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM K5 analogue dilutedinto 16 ml of 20 mM sodium phosphate buffer (pH 7.0), 5 mM sodiumcaprylate and 750 mM (NH₄)₂SO₄ was performed on a column of Butylsepharose using the gradient #1 described above. In FIG. 6 the purifiedconjugate fraction appears in fraction A with albumin and in fraction B(F6-F7-F8).

Example 7 Purification of HSA:First Insulin Derivative (SEQ ID NO:4)Conjugate

The first insulin derivative is human insulin with MPA on position B1and is represented in FIG. 1 below.

The purification of a conjugate made from reacting 1 ml 25% 250 mg/mlHSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM first insulinderivative diluted into 9 ml of 20 mM sodium phosphate buffer (pH 7.0),5 mM sodium caprylate and 750 mM (NH₄)₂SO₄ was performed on a column ofButyl sepharose using the gradient #1 described above. In FIG. 7 thepurified conjugate fraction appears in fraction B (F6-F7-F8).

Example 8 Purification of HSA:Second Insulin Derivative (SEQ ID NO:5)Conjugate

The second insulin derivative is human insulin with MPA on position A1and is represented in FIG. 1 shown above in Example 7.

The purification of a conjugate made from reacting 1 ml 25% 250 mg/mlHSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM second insulinderivative diluted into 9 ml of 20 mM sodium phosphate buffer (pH 7.0),5 mM sodium caprylate and 750 mM (NH₄)₂SO₄ was performed on a column ofButyl sepharose using gradient #1 described above. In FIG. 8 thepurified conjugate fraction appears in fraction B (F6-F7-F8).

Example 9 Purification of HSA:First C34 Analogue (SEQ ID NO:6) Conjugate

The first C34 analogue is MPA-AEEA-C34-CONH₂ and has the followingsequence:

The purification of a conjugate made from reacting 5 ml 25% 250 mg/mlHSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM first C34 analoguediluted into 20 ml of 20 mM sodium phosphate buffer (pH 7.0), 5 mMsodium caprylate and 750 mM (NH₄)₂SO₄ was performed on a column of Butylsepharose using gradient #1 described above. In FIG. 9 the purifiedconjugate fraction appears in fraction F2.

Example 10 Purification of HSA:Second C34 Analogue (SEQ ID NO:7)Conjugate

The second C34 analogue is C34 (1-34) Lys³⁵ (ε-AEEA-MPA)-CONH₂ and hasthe following structure:

The purification of a conjugate made from reacting 5 ml 25% 250 mg/mlHSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM second C34 analoguediluted into 20 ml of 20 mM sodium phosphate buffer, 5 mM sodiumcaprylate and 750 mM (NH₄)₂SO₄ was performed on a column of Butylsepharose using gradient #1 described above. In FIG. 10 the purifiedconjugate fraction appears in fraction F2.

Example 11 Purification of HSA:Third C34 Analogue (SEQ ID NO:8)Conjugate

The third C34 analogue is C34 (1-34) Lys¹³ (ε-AEEA-MPA)-CONH₂ and hasthe following structure:

The purification of a conjugate made from reacting 5 ml 25% 250 mg/mlHSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM third C34 analoguediluted into 20 ml of 20 mM sodium phosphate buffer (pH 7.0), 5 mMsodium caprylate and 750 mM (NH₄)₂SO₄, was performed on a column ofButyl sepharose using gradient #1 described above. In FIG. 11 thepurified conjugate fraction appears in fraction F2.

Example 12 Purification of l-Cysteine

The purification of 121 mg of 1-cysteine in 2 ml of a buffer made of 20mM sodium phosphate, 5 mM sodium caprylate and 750 mM (NH₄)₂SO₄, wasperformed on a column of Butyl sepharose using the gradient #5 describedabove. FIG. 12 illustrates the separation curve obtained, whereL-cysteine elutes within the void volume of the column (F3).

Example 13 Purification of L-cysteine:First GLP-1 Analogue (SEQ ID NO:1)Conjugate

The first GLP-1 analogue is GLP-1 (7-36) dAla⁸ Lys³⁷ (ε-AEEA-MPA)-CONH₂and his sequence is shown above in Example 1.

The purification of a conjugate made from reacting 121 mg L-cysteinewith 36.36 mg first GLP-1 analogue diluted into 2 ml of 20 mM sodiumphosphate buffer (pH 7.0), 5 mM sodium caprylate and 750 mM (NH₄)₂SO₄was performed on a column of Butyl sepharose using gradient #5 describedabove. FIG. 13 illustrates the separation curve obtained where theexcess L-cysteine elutes in F3 (column void volume) and theL-Cysteine:first GLP-1 analogue conjugate elutes in 0 mM (NH₄)₂SO₄.

Example 14 Purification of HSA:Second GLP-1 Analogue (SEQ ID NO:9)Conjugate

The second GLP-1 analogue is GLP-1 (7-36) Lys³⁷ (ε-MPA)-NH₂ and has thefollowing sequence:

HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRK(ε-MPA)

The purification of a conjugate made from reacting 2.5 ml 25% 250 mg/mlHSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM second GLP-1analogue diluted into 10 ml of 20 mM sodium phosphate buffer (pH 7.0), 5mM sodium caprylate and 750 mM (NH₄)₂SO₄ was performed on a column ofButyl sepharose using gradient #5 described above. In FIG. 14 thepurified conjugate fraction appears in fraction F2.

Example 15 Purification of HSA:Third GLP-1 Analogue (SEQ ID NO:10)Conjugate

The third GLP-1 analogue is GLP-1 (7-36) dAla⁸ Lys³⁷ (ε-MPA)-NH₂ and hasthe following sequence:

H(dA)EGTFTSDVSSYLEGQAAKEFIAWLVKGRK(MPA)-CONH₂

The purification of a conjugate made from reacting 2.5 ml 25% 250 mg/mlHSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM third GLP-1 analoguediluted into 10 ml of 20 mM sodium phosphate buffer (pH 7.0), 5 mMsodium caprylate and 750 mM (NH₄)₂SO₄ was performed on a column of Butylsepharose using gradient #5 described above. In FIG. 15 the purifiedconjugate fraction appears in fraction F2.

Example 16 Purification of HSA:Fourth GLP-1 Analogue (SEQ ID NO:11)Conjugate

The fourth GLP-1 analogue is GLP-1 (7-36) Lys²⁶ (ε-AEEA-AEEA-MPA) andhas the following sequence:

HAEGTFTSDVSSYLEGQMK(ε-AEEA-AEEA-MPA) EFIAWLVKGR

The purification of a conjugate made from reacting 2.5 ml 25% 250 mg/mlHSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM fourth GLP-1analogue diluted into 10 ml of 20 mM sodium phosphate buffer (pH 7.0), 5mM sodium caprylate and 750 mM (NH₄)₂SO₄ was performed on a column ofButyl sepharose using gradient #1 described above. In FIG. 16 thepurified conjugate fraction appears in fraction F2.

Example 17 Purification of HSA:Fifth GLP-1 Analogue (SEQ ID NO:12)Conjugate

The fifth GLP-1 analogue is GLP-1 (7-36) Lys³⁴ (ε-AEEA-AEEA-MPA) and hasthe following sequence:

HAEGTFTSDVSSYLEGQAAKEFIAWLVK(ε-AEEA-AEEA-MPA)GR

The purification of a conjugate made from reacting 2.5 ml 25% 250 mg/mlHSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM fifth GLP-1 analoguediluted into 10 ml of 20 mM sodium phosphate buffer (pH 7.0), 5 mMsodium caprylate and 750 mM (NH₄)₂SO₄ was performed on a column of Butylsepharose using gradient #1 described above. In FIG. 17 the purifiedconjugate fraction appears in fraction F2.

Example 18 Purification of HSA:First Exendin-4 Analogue (SEQ ID NO:13)Conjugate

The first exendin-4 analogue is Exendin-4-(1-39) Lys⁴⁰ (ε-MPA)-NH₂ andhas the following sequence:

HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSK(ε-MPA)- CONH₂

The purification of a conjugate made from reacting 1 ml 25% 250 mg/mlHSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM first Exendin-4analogue diluted into 9 ml of 20 mM sodium phosphate buffer (pH 7.0), 5mM sodium caprylate and 750 mM (NH₄)₂SO₄ was performed on a column ofButyl sepharose using gradient #1 described above. In FIG. 18 thepurified conjugate fraction appears in fraction F2.

Example 19 Purification of HSA:Second Exendin-4 Analogue (SEQ ID NO:14)Conjugate

The second Exendin-4 analogue is Exendin-4 (9-39) Lys⁴⁰(ε-AEEA-MPA)-CONH₂ and has the following sequence:

DLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSK(AEEA-MPA)-CONH₂

The purification of a conjugate made from reacting 3.5 ml 25% HSA cortexwith 1 mM second Exendin-4 analogue diluted into 21.5 ml of 20 mM sodiumphosphate buffer (pH 7.0), 5 mM sodium caprylate and 750 mM (NH₄)₂SO₄was performed on a column of Butyl sepharose using gradient #1 describedabove. In FIG. 19 the purified conjugate fraction appears in fractionF2.

Example 20 Purification of HSA:MPA

The purification of a conjugate made from reacting 1 ml 25% 250 mg/mlHSA (Cortex-Biochem, San Leandro, Calif.) with 2 mM MPA diluted into 9ml of 20 mM sodium phosphate buffer (pH 7.0), 5 mM sodium caprylate and1750 mM (NH₄)₂SO₄ was performed on a column of Butyl sepharose usinggradient #2 described above. In FIG. 20 the fraction of mercaptalbuminis in fraction A (F5) and capped albumin is in fraction B (F7-F8). Theconjugate fraction was concentrated with Amicon™ filter 30 kDa.

Example 21 Purification of HSA

The purification of 1 ml 25% 250 mg/ml HSA (Cortex-Biochem, San Leandro,Calif.) diluted into 9 ml of 20 mM sodium phosphate buffer (pH 7.0), 5mM sodium caprylate and 1750 mM (NH₄)₂SO₄ was performed on a column ofButyl sepharose using the gradient #2 described above. When usinggradient #2, unlike gradients #1 and #5, both conjugated albumin andnon-conjugated albumin adsorbs onto the hydrophobic resin during sampleloading. FIG. 21 illustrates the separation curve obtained where F4 andF5 are enriched in mercaptalbumin and F6, F7 and F8 are enriched incapped albumin.

Example 22 Purification of HSA:Second C34 Analogue (SEQ ID NO:3)Conjugate

The second C34 analogue is C34 (1-34) Lys³⁵ (ε-AEEA-MPA)-CONH₂ and hisstructure is shown in Example 10.

The purification of a conjugate made from reacting 1 ml 25% 250 mg/mlHSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM second C34 analoguediluted into 9 ml of a buffer made of 20 mM sodium phosphate buffer (pH7.0), 5 mM sodium caprylate and 1750 mM (NH₄)₂SO₄ was performed on acolumn of Butyl sepharose using gradient #2 described above. In FIG. 22mercaptalbumin appears in fraction A (F5) and capped albumin and thepurified conjugated is in fraction B (F7-F8).

Example 23 Purification of HSA:First Dynorphin A Analogue (SEQ ID NO:15)Conjugate

The first Dynorphin A analogue is Dyn A (1-13) (MPA)-NH₂ and has thefollowing sequence:

YGGFLRRIRPKLK(MPA)-CONH₂.

The purification of a conjugate made from reacting 1 ml 25% 250 mg/mlHSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM first Dynorphin Aanalogue diluted into 9 ml of 20 mM sodium phosphate buffer (pH 7.0), 5mM sodium caprylate and 1750 mM (NH₄)₂SO₄ was performed on a column ofButyl sepharose using gradient #2 described above. In FIG. 23 thepurified conjugate fraction appears in fraction A (F11-F12)

Example 24 Purification of HSA:First ANP Analogue (SEQ ID NO:16)Conjugate

The first ANP analogue is MPA-AEEA-ANP (99-126)-CONH₂ and has thefollowing structure:

The purification of a conjugate made from reacting 1 ml 25% 250 mg/mlHSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM first ANP analoguediluted into 9 ml of 20 mM sodium phosphate buffer (pH 7.0), 5 mM sodiumcaprylate and 1750 mM (NH₄)₂SO₄ was performed on a column of Butylsepharose using gradient #2 described above. In FIG. 24 the purifiedconjugate fraction appears in fraction A (F14).

Example 25 Purification of HSA:Second Dynorphin A Analogue (SEQ IDNO:17) Conjugate

The second Dynorphin A analogue is Dyn A (7-13) Lys¹³ (ε-MPA)-CONH₂ andhas the following sequence:

RIRPKLK(MPA)-CONH₂

The purification of a conjugate made from reacting 1 ml 25% 250 mg/mlHSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM second Dynorphin Aanalogue diluted into 9 ml of 20 mM sodium phosphate buffer (pH 7.0), 5mM sodium caprylate and 1750 mM (NH₄)₂SO₄ was performed on a column ofButyl sepharose using gradient #2 described above. In FIG. 25 thepurified conjugate fraction appears in fraction A (F9).

Example 26 Purification of HSA:ACE Inhibitor (SEQ ID NO:18) Conjugate

The ACE inhibitor used in this example isacetyl-Phe-His-cyclohexylstatyl-Ile-Lys (ε-AEEA-MPA)-CONH₂ and has thefollowing sequence:

The purification of a conjugate made from reacting 1 ml 25% 250 mg/mlHSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM ACE inhibitordiluted into 9 ml of 20 mM sodium phosphate buffer (pH 7.0), 5 mM sodiumcaprylate and 1750 mM (NH₄)₂SO₄ was performed on a column of Butylsepharose using gradient #2 described above. In FIG. 26 the purifiedconjugate fraction appears in fraction A (F14).

Example 27 Purification of HSA:Sixth GLP-1 Analogue (SEQ ID NO:19)Conjugate

The sixth GLP-1 analogue is GLP-1 (7-36) Lys²³ (ε-AEEA-MPA)-CONH₂ andhas the following sequence:

HAEGTFTSDVSSYLEGK(AEEA-MPA)AAKEFIAWLVKGR-CONH₂

The purification of a conjugate made from reacting 3 ml 25% 250 mg/mlHSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM sixth GLP-1 analoguediluted into 22 ml of 20 mM sodium phosphate buffer (pH 7.0), 5 mMsodium caprylate and 1750 mM (NH₄)₂SO₄ was performed on a column ofButyl sepharose using gradient #1 described above. In FIG. 27 thepurified conjugate fraction appears in fraction F2.

Example 28 Purification of HSA:Seventh GLP-1 Analogue (SEQ ID NO:20)Conjugate

The seventh GLP-1 analogue is GLP-1 (7-36) Lys¹⁸ (ε-AEEA-MPA)-CONH₂ andhas the following sequence:

HAEGTFTSDVSK(AEEA-MPA)YLEGQAAKEFIAWLVKGR-CONH₂

The purification of a conjugate made from reacting 3 ml 25% 250 mg/mlHSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM seventh GLP-1analogue diluted into 22 ml of 20 mM sodium phosphate buffer (pH 7.0), 5mM sodium caprylate and 750 mM (NH₄)₂SO₄ was performed on a column ofButyl sepharose using gradient #1 described above. In FIG. 28 thepurified conjugate fraction appears in fraction F2.

Example 29 Purification of HSA:Eighth GLP-1 Analogue (SEQ ID NO:21)Conjugate

The eighth GLP-1 analogue is GLP-1 (7-36) Lys²⁶ (ε-AEEA-MPA)-CONH₂ andhas the following sequence:

HAEGTFTSDVSSYLEGQAAK(AEEA-MPA)EFIAWLVKGR-CONH₂

The purification of a conjugate made from reacting 2.5 ml 25% 250 mg/mlHSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM eighth GLP-1analogue diluted into 22.5 ml of 20 mM sodium phosphate buffer (pH 7.0),5 mM sodium caprylate and 750 mM (NH₄)₂SO₄ was performed on a column ofButyl sepharose using gradient #1 described above. In FIG. 29 thepurified conjugate fraction appears in fraction F2.

Example 30 Purification of HSA:Ninth GLP-1 Analogue (SEQ ID NO:22)Conjugate

The ninth GLP-1 analogue is GLP-1 (7-37) Lys²⁷ (ε-AEEA-MPA)-CONH₂ andhas the following sequence:

HAEGTFTSDVSSYLEGQAAKK(AEEA-MPA)FIAWLVKGR-CONH₂

The purification of a conjugate made from reacting 3 ml 25% 250 mg/mlHSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM ninth GLP-1 analoguediluted into 22 ml of 20 mM sodium phosphate buffer (pH 7.0), 5 mMsodium caprylate and 750 mM (NH₄)₂SO₄ was performed on a column of Butylsepharose using gradient #1 described above. In FIG. 30 the purifiedconjugate fraction appears in fraction F2.

Example 31 Purification of HSA:Tenth GLP-1 Analogue (SEQ ID NO:23)Conjugate

The tenth GLP-1 analogue is GLP-1 (7-36) Lys³⁷ (ε-AEEA-AEEA-MPA)-CONH₂and has the following sequence:

HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRK-AEEA-AEEA-MPA- CONH₂

The purification of a conjugate made from reacting 2.5 ml 25% 250 mg/mlHSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM tenth GLP-1 analoguediluted into 22.5 ml of 20 mM sodium phosphate buffer (pH 7.0), 5 mMsodium caprylate and 750 mM (NH₄)₂SO₄ was performed on a column of Butylsepharose using gradient #1 described above. In FIG. 31 the purifiedconjugate fraction appears in fraction F2.

Example 32 Purification of HSA:Eleventh GLP-1 Analogue (SEQ ID NO:24)Conjugate

The eleventh GLP-1 analogue is GLP-1 (7-36) Lys³⁷ (ε-AEEA-MPA)-CONH₂ andhas the following sequence:

HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRK(AEEA-MPA)-CONH₂

The purification of a conjugate made from reacting 2.5 ml 25% 250 mg/mlHSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM eleventh GLP-1analogue diluted into 22.5 ml of 20 mM sodium phosphate buffer (pH 7.0),5 mM sodium caprylate and 750 mM (NH₄)₂SO₄ was performed on a column ofButyl sepharose using gradient #1 described above. In FIG. 32 thepurified conjugate fraction appears in fraction F2.

Example 33 Purification of HSA:Third Exendin-4 Analogue (SEQ ID NO:25)Conjugate

The third Exendin-4 analogue is Exendin-4-(1-39) Lys⁴⁰(ε-AEEA-MPA)-CONH₂ and has the following sequence:

HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSK(ε-AEEA- MPA)-CONH₂

The purification of a conjugate made from reacting 2.5 ml 25% 250 mg/mlHSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM third Exendin-4analogue diluted into 22.5 ml of 20 mM sodium phosphate buffer (pH 7.0),5 mM sodium caprylate and 750 mM (NH₄)₂SO₄ was performed on a column ofButyl sepharose using gradient #1 described above. In FIG. 33 thepurified conjugate fraction appears in fraction F2.

Example 34 Purification of HSA:Twelfth GLP-1 Analogue (SEQ ID NO:26)Conjugate

The twelfth GLP-1 analogue is GLP-1 (7-36) Lys³⁴ (ε-AEEA-MPA)-CONH₂ andhas the following sequence:

HAEGTFTSDVSSYLEGQAAKEFIAWLVK(ε-AEEA-MPA)GR-CONH₂

The purification of a conjugate made from reacting 2.5 ml 25% 250 mg/mlHSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM twelfth GLP-1analogue diluted into 22.5 ml of 20 mM sodium phosphate buffer (pH 7.0),5 mM sodium caprylate and 750 mM (NH₄)₂SO₄ was performed on a column ofButyl sepharose using gradient #1 described above. In FIG. 34 thepurified conjugate fraction appears in fraction F2.

Example 35 Purification of HSA:First Insulin Derivative (SEQ ID NO:4)Conjugate

The first insulin derivative is human insulin with MPA on position B1and his structure is detailed in Example 7.

The purification of a conjugate made from reacting 2.5 ml 25% 250 mg/mlHSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM first insulinderivative diluted into 22.5 ml of 20 mM sodium phosphate buffer (pH7.0), 5 mM sodium caprylate and 750 mM (NH₄)₂SO₄ was performed on acolumn of Butyl sepharose using gradient #1 described above. In FIG. 35the purified conjugate fraction appears in fraction F2.

Example 36 Purification of HSA:Third Insulin Derivative (SEQ ID NO:27)Conjugate

The third insulin derivative is human insulin with OA-MPA on position B1and is represented in FIG. 1 shown above in Example 7.

The purification of a conjugate made from reacting 4 ml 25% 250 mg/mlHSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM third insulinderivative diluted into 21 ml of 20 mM sodium phosphate buffer (pH 7.0),5 mM sodium caprylate and 750 mM (NH₄)₂SO₄ was performed on a column ofButyl sepharose using gradient #1 described above. In FIG. 36 thepurified conjugate fraction appears in fraction F2.

Example 37 Purification of HSA:Second Insulin Derivative (SEQ ID NO:5)Conjugate

The second insulin derivative is human insulin with MPA on position A1and is represented in FIG. 1 shown above in Example 7.

The purification of a conjugate made from reacting 3 ml 25% 250 mg/mlHSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM second insulinderivative diluted into 22 ml of 20 mM sodium phosphate buffer (pH 7.0),5 mM sodium caprylate and 750 mM (NH₄)₂SO₄ was performed on a column ofButyl sepharose using gradient #1 described above. In FIG. 37 thepurified conjugate fraction appears in fraction F2.

Example 38 Purification of HSA:Fourth Insulin Derivative (SEQ ID NO:28)Conjugate

The fourth insulin derivative is human insulin with MPA on position B29and is represented in FIG. 1 shown above in Example 7.

The purification of a conjugate made from reacting 3 ml 25% 250 mg/mlHSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM fourth insulinderivative diluted into 22 ml of 20 mM sodium phosphate buffer (pH 7.0),5 mM sodium caprylate and 750 mM (NH₄)₂SO₄ was performed on a column ofButyl sepharose using gradient #1 described above. In FIG. 38 thepurified conjugate fraction appears in fraction F2.

Example 39 Purification of HSA:First GRF Analogue (SEQ ID NO:2)Conjugate

The first GRF analogue is GRF (1-29) dAla² Gln⁸ Ala¹⁵ Leu²⁷ Lys³⁰(ε-MPA) CONH₂ and his sequence is shown in Example 2.

The purification of a conjugate made from reacting 3.7 ml 25% 250 mg/mlHSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM first GRF analoguediluted into 22 ml of 20 mM sodium phosphate buffer (pH 7.0), 5 mMsodium caprylate and 750 mM (NH₄)₂SO₄ was performed on a column of Butylsepharose using gradient #1 described above. In FIG. 39 the purifiedconjugate fraction appears in fraction F2.

Example 40 Purification of HSA:Second GRF Analogue (SEQ ID NO:29)Conjugate

The second GRF analogue is GRF(1-29) Lys³⁰ (ε-MPA)-CONH₂ and has thefollowing sequence:

YADAIFTNSYRKVLGQLSARKLLQDIMSRK(MPA)-CONH₂

The purification of a conjugate made from reacting 2.5 ml 25% 250 mg/mlHSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM second GRF analoguediluted into 22.5 ml of 20 mM sodium phosphate buffer (pH 7.0), 5 mMsodium caprylate and 900 mM (NH₄)₂SO₄ was performed on a column of Butylsepharose using gradient #3 described above. In FIG. 40 the purifiedconjugate fraction appears in fraction F2.

Example 41 Purification of HSA:Third GRF Analogue (SEQ ID NO:30)Conjugate

The third GRF analogue is GRF (1-29) dAla² Gln⁸ dArg¹¹ Ala¹⁵ Leu²⁷ Lys³⁰(ε-MPA)-CONH₂ and has the following sequence:

YaDAIFTQSYrKVLAQLSARKLLQDILSRK(MPA)-CONH₂

The purification of a conjugate made from reacting 2.5 ml 25% 250 mg/mlHSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM third GRF analoguediluted into 22.5 ml of 20 mM sodium phosphate buffer (pH 7.0), 5 mMsodium caprylate and 750 mM (NH₄)₂SO₄ was performed on a column of Butylsepharose using gradient #3 described above. In FIG. 41 the purifiedconjugate fraction appears in fraction F2.

Example 42 Purification of HSA:Fourth GRF Analogue (SEQ ID NO:31)Conjugate

The fourth GRF analogue is GRF (1-29) dAla² Lys³⁰ (ε-MPA)-CONH₂ and hasthe following sequence:

YaDAIFTNSYRKVLGQLSARKLLQDIMSRK(MPA)-CONH₂

The purification of a conjugate made from reacting 2.5 ml 25% HSA(Cortex-Biochem, San Leandro, Calif.) with 1 mM fourth GRF analoguediluted in 22.5 ml of 20 mM sodium phosphate buffer (pH 7.0), 5 mMsodium caprylate and 900 mM (NH₄)₂SO₄ was performed on a column of Butylsepharose using gradient #3 described above. In FIG. 42 the purifiedconjugate fraction appears in fraction F2.

Example 43 Purification of HSA:Thirteenth GLP-1 Analogue CJC 1365 (SEQID NO:32) Conjugate

The thirteenth GLP-1 analogue is GLP-1 (9-36) Lys³⁷ (ε-AEEA-MPA)-CONH₂and has the following sequence:

EGTFTSDVSSYLEGQAAKEFIAWLVKGRK(ε-AEEA-MPA)-CONH₂

The purification of a conjugate made from reacting 3.5 ml 25% HSA(Cortex-Biochem, San Leandro, Calif.) and 1 mM thirteenth GLP-1 analoguediluted in 21.5 ml of 20 mM sodium phosphate buffer (pH 7.0), 5 mMsodium caprylate and 750 mM (NH₄)₂SO₄ was performed on a column of Butylsepharose using gradient #1 described above. In FIG. 43 the purifiedconjugate fraction appears in fraction F2.

Example 44 Purification of HSA Lactose:First GLP-1 Analogue (SEQ IDNO:1) Conjugate

The first GLP-1 analogue is GLP-1 (7-36) dAla⁸ Lys³⁷ (ε-AEEA-MPA)-CONH₂and his sequence is shown above in Example 1.

The purification of a conjugate made from reacting 4 ml 25%lactosaminated albumin (HSA pre-incubated with excess lactose at 37° C.,pH 7.0) with 200 μM first GLP-1 analogue in 4 ml of a buffer made of 20mM sodium phosphate, 5 mM sodium caprylate and 750 mM (NH₄)₂SO₄, (pH7.0) was performed on a column of Butyl sepharose using gradient #1described above. In FIG. 44 the purified lactosaminated conjugatefraction appears in fraction F2.

Example 45 Purification of HSA:First T20 Analogue (SEQ ID NO:33)Conjugate

The first T20 analogue is Ac-T20 (1-36) Lys³⁷ (ε-AEEA-MPA)-CONH₂ and ahsthe following sequence:

Ac-YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFK(AEEA- MPA)-CONH₂

The purification of a conjugate made from reacting 2.5 ml 25% HSA with 1mM first T20 analogue in 10 ml of 20 mM sodium phosphate buffer (pH7.0), 5 mM sodium caprylate and 750 mM (NH₄)₂SO₄ was performed on acolumn of Butyl sepharose using gradient #1 described above. In FIG. 45the purified conjugate fraction appears in fraction F2.

Example 46 Purification of HSA:First T1249 Analogue (SEQ ID NO:34)Conjugate

The first T1249 analogue is Ac-T1249 (1-39) Lys⁴⁰ (ε-AEEA-MPA)-CONH₂ andhas the following sequence:

Ac-WQEWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWFK(AEEA- MPA)-CONH₂

The purification of a conjugate made from reacting 2 ml 25% HSA and 1 mMfirst T1249 analogue in 10.5 ml of 20 mM sodium phosphate buffer (pH7.0), 5 mM sodium caprylate and 750 mM (NH₄)₂SO₄ was performed on acolumn of Butyl sepharose using gradient #1 described above. In FIG. 46the purified conjugate fraction appears in fraction F4.

Example 47 Purification of a HSA:First GLP-1 Analogue (SEQ ID NO:1)

The first GLP-1 analogue is GLP-1 (7-36) dAla⁸ Lys³⁷ (ε-AEEA-MPA)-CONH₂and his sequence is shown in Example 1.

The purification of 114.45 mg of the preformed conjugate of the firstGLP-1 analogue in 12.5 ml of 20 mM sodium phosphate buffer (pH 7.0), 5mM sodium caprylate and 750 mM (NH₄)₂SO₄ was performed on a column ofButyl sepharose using gradient #5 described above. FIG. 47 illustratesthe separation curve obtained with the conjugate found in fraction F2.

Example 48 Purification of a HSA:First C34 Analogue (SEQ ID NO:6)

The first C34 analogue is MPA-AEEA-C34-CONH₂ and his sequence is shownabove in Example 9.

The purification of 114.45 mg of the preformed conjugate of the firstC34 analogue in 12.5 ml of 20 mM sodium phosphate buffer (pH 7.0), 5 mMsodium caprylate and 750 mM (NH₄)₂SO₄ was performed on a column of Butylsepharose using gradient #5 described above. FIG. 48 illustrates theseparation curve obtained with the conjugate found in fraction F2.

Example 49 Purification of a HSA:Second GRF Analogue (SEQ ID NO:29)

The second GRF analogue is GRF(1-29) Lys³⁰ (ε-MPA)-CONH₂ and hissequence is shown above in Example 40.

The purification of 125.53 mg of the preformed conjugate of the secondGRF analogue in 12.5 ml of 20 mM sodium phosphate buffer (pH 7.0), 5 mMsodium caprylate and 750 mM (NH₄)₂SO₄, pH 7.0 was performed on a columnof Butyl sepharose using gradient #5 described above. FIG. 49illustrates the separation curve obtained with the conjugate found infraction F2.

Example 50 Purification of HSA:Vinorelbine Analogue Conjugate (SEQ IDNO:35)

The vinorelbine analogue is a molecule of vinorelbine with AEEA-MPAcoupled thereto as illustrated in the following structure:

The purification of a conjugate made from 2.5 ml 25% HSA and 1 mMvinorelbine analogue in 22.5 ml of a buffer made of 20 mM sodiumphosphate buffer, 5 mM sodium caprylate and 750 mM (NH₄)₂SO₄, pH 7.0 wasperformed on a column of Butyl sepharose using gradient #4 describedabove. In FIG. 50 the purified conjugate fraction appears in fractionF2. The conjugate fraction was concentrated with Amicon™ filter 30 kDa.

Example 51 Purification of L-Cysteine

The purification of 2.5 ml 40 mM L-cysteine in 22.5 ml of 20 mM sodiumphosphate buffer (pH 7.0), 5 mM sodium caprylate and 1500 mM (NH₄)₂SO₄,was performed on a column of Butyl sepharose using gradient #4 describedabove. FIG. 51 illustrates the separation curve obtained with L-cysteineeluting within the void volume of the column (fraction F3).

Example 52 Purification of L-Cysteine:Vinorelbine Analogue (SEQ IDNO:35) Conjugate

The vinorelbine analogue is a molecule of vinorelbine with AEEA-MPAcoupled thereto as illustrated in the structure shown in Example 50.

The purification of a conjugate made from reacting 2.5 ml 40 mML-cysteine with 1 mM vinorelbine analogue in 22.5 ml of 20 mM sodiumphosphate buffer (pH 7.0), 5 mM sodium caprylate and 750 mM (NH₄)₂SO₄was performed on a column of Butyl sepharose using gradient #4 describedabove. FIG. 52 illustrates the separation curve obtained with theL-cysteine conjugate eluting within fractions F8, F9 and F10.

Example 53 Purification of RSA:Third Exendin-4 Analogue (SEQ ID NO:25)Conjugate

The third Exendin-4 analogue is Exendin-4-(1-39) Lys⁴⁰(ε-AEEA-MPA)-CONH₂ and his sequence shown in Example 33.

The purification of a conjugate made from reacting 11 ml 5% RSA (ratserum albumin) with 200 μM third Exendin-4 analogue in 11 ml of 20 mMsodium phosphate buffer (pH 7.0), 5 mM sodium caprylate and 750 mM(NH₄)₂SO₄, was performed on a column of Butyl sepharose using gradient#1 described above. In FIG. 53 the purified conjugate fraction appearsin fraction F2.

Example 54 Purification of HSA:Fourth C34 Analogue (SEQ ID NO:36)Conjugate

The fourth C34 analogue is C34 (1-34) Lys¹³ (ε-MPA)-CONH₂ and has thefollowing sequence:

WMEWDREINNYTK(MPA)LIHSLIEESQNQQEKNEQELL-CONH₂

The purification of a conjugate made from reacting 2 ml 25% HSA with 1mM fourth C34 analogue in 13 ml of 20 mM sodium phosphate buffer (pH7.0), 5 mM sodium caprylate and 750 mM (NH₄)₂SO₄ was performed on acolumn of Butyl sepharose using gradient #1 described above. In FIG. 54the purified conjugate fraction appears in fraction F2.

Example 55 Purification of HSA:Fifth C34 Analogue (SEQ ID NO:37)Conjugate

The fifth C34 analogue is C34 (1-34) Lys³⁵ (ε-MPA)-CONH₂ and has thefollowing sequence:

WMEWDREINNYTSLIHSLIEESQNQQEKNEQELLK(MPA)-CONH₂

The purification of a conjugate made from 2 ml 25% HSA and 1 mM fifthC34 analogue in 13 ml of a buffer made of 20 mM sodium phosphate buffer(pH 7.0), 5 mM sodium caprylate and 750 mM (NH₄)₂SO₄, was performed on acolumn of Butyl sepharose using gradient #1 described above. In FIG. 55the purified conjugate fraction appears in fraction F2.

Example 56 Purification of HSA:Sixth C34 Analogue (SEQ ID NO:38)Conjugate

The sixth C34 analogue MPA-C34 (1-34)-CONH₂ and has the followingsequence:

MPA-WMEWDREINNYTSLIHSLIEESQNQQEKNEQELL-CONH₂

The purification of a conjugate made from reacting 2 ml 25% HSA and 1 mMsixth C34 analogue in 13 ml of 20 mM sodium phosphate buffer (pH 7.0), 5mM sodium caprylate and 750 mM (NH₄)₂SO₄ was performed on a column ofButyl sepharose using gradient #1 described above. In FIG. 56 thepurified conjugate fraction appears in fraction F2.

Example 57 Purification of HSA:Seventh C34 Analogue (SEQ ID NO:39)Conjugate

The seventh C34 analogue is Ac-C34 (1-34) Glu² Lys⁶ Lys⁷ Glu⁹ Glu¹⁰Lys¹³ Lys¹⁴ Glu¹⁶ Glu¹⁷ Lys²⁰ Lys²¹ Glu²³ Glu²⁴ Lys²⁷ Glu³¹ Lys³⁴ Lys³⁵Lys³⁶ (ε-AEEA-MPA)-CONH₂ and has the following sequence:

Ac-WEEWOKKIEEYTKKIEELIKKSEEQQKKNEEELKKK(AEEA-MPA)- CONH₂

The purification of a conjugate made from reacting 2 ml 25% HSA with 1mM seventh C34 analogue in 13 ml 20 mM sodium phosphate buffer (pH 7.0),5 mM sodium caprylate and 750 mM (NH₄)₂SO₄, was performed on a column ofButyl sepharose using gradient #1 described above. In FIG. 57 thepurified conjugate fraction appears in fraction F2.

Example 58 Purification of HSA:Eighth C34 Analogue (SEQ ID NO:40)Conjugate

The eighth C34 analogue is MPA-AEEA-C34 (1-34) Glu² Lys⁶ Lys⁷ Glu⁹ Glu¹⁰Lys¹³ Lys¹⁴ Glu¹⁶ Glu¹⁷ Lys²⁰ Lys²¹ Glu²³ Glu²⁴ Lys²⁷ Glu³¹ Lys³⁴Lys³⁵-CONH₂ and has the following sequence:

MPA-AEEA-WEEWDKKIEEYTKKIEELIKKSEEQQKKNEEELKK-CONH₂

The purification of a conjugate made from reacting 2 ml 25% HSA with 1mM eighth C34 analogue in 13 ml of 20 mM sodium phosphate buffer (pH7.0), 5 mM sodium caprylate and 750 mM (NH₄)₂SO₄, was performed on acolumn of Butyl sepharose using gradient #1 described above. In FIG. 58the purified conjugate fraction appears in fraction F2.

Example 59 Purification of HSA:First PYY Analogue (SEQ ID NO:41)Conjugate

The first PYY analogue is PYY (3-36) Lys⁴ (ε-OA-MPA)-CONH₂ and has thefollowing structure:

The purification of a conjugate made from reacting 1.5 ml 25% HSA with 1mM first PYY analogue in 6 ml of 20 mM sodium phosphate buffer (pH 7.0),5 mM sodium caprylate and 750 mM (NH₄)₂SO₄, was performed on a column ofButyl sepharose using gradient #1 described above. In FIG. 59 thepurified conjugate fraction appears in fraction F2.

Example 60 Purification of HSA:Second PYY Analogue (SEQ ID NO:42)Conjugate

The second PYY analogue is MPA-OA-PYY (3-36)-CONH₂ and has the followingsequence:

The purification of a conjugate made from reacting 1.5 ml 25% HSA with 1mM second PYY analogue in 6 ml of 20 mM sodium phosphate buffer (pH7.0), 5 mM sodium caprylate and 750 mM (NH₄)₂SO₄, was performed on acolumn of Butyl sepharose using gradient #1 described above. In FIG. 60the purified conjugate fraction appears in fraction F2.

Example 61 Purification of HSA:Fifth Insulin Derivative (SEQ ID NO:43)Conjugate

The fifth insulin derivative is human insulin with AEEAS-AEEAS-MPA onposition B29 and is represented in FIG. 1 shown above in Example 7.

The purification of a conjugate made from reacting 2 ml 25% HSA with 1mM fifth insulin derivative in 15 ml of 20 mM sodium phosphate buffer(pH 7.0), 5 mM sodium caprylate and 750 mM (NH₄)₂SO₄ was performed on acolumn of Butyl sepharose using gradient #1 described above. In FIG. 61the purified conjugate fraction appears in fraction F2.

Example 62 Purification of HSA:Sixth Insulin Derivative (SEQ ID NO:44)Conjugate

The sixth insulin derivative is human insulin with AEEAS-AEEAS-MPA onposition B1 and is represented in FIG. 1 shown above in Example 7.

The purification of a conjugate made from reacting 2.5 ml 25% HSA with 1mM sixth insulin derivative in 15 ml of 20 mM sodium phosphate buffer(pH 7.0), 5 mM sodium caprylate and 750 mM (NH₄)₂SO₄ was performed on acolumn of Butyl sepharose using gradient #1 described above. In FIG. 62the purified conjugate fraction appears in fraction F2.

Example 63 Purification of HSA:Seventh Insulin Derivative (SEQ ID NO:45)Conjugate

The seventh insulin derivative is human insulin with OA-MPA on positionB29 and is represented in FIG. 1 shown above in Example 7.

The purification of a conjugate made from reacting 2 ml 25% HSA with 1mM seventh insulin derivative in 15 ml of 20 mM sodium phosphate buffer(pH 7.0), 5 mM sodium caprylate and 750 mM (NH₄)₂SO₄ was performed on acolumn of Butyl sepharose using gradient #1 described above. In FIG. 63the purified conjugate fraction appears in fraction F2.

Example 64 Purification of HSA:Third PYY Analogue (SEQ ID NO:46)Conjugate

The third PYY analogue is MPA-PYY (3-36)-CONH₂ and has the followingsequence:

MPA-NH-IKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY-CONH₂

The purification of a conjugate made from reacting 3 ml 25% HSA with 1mM third PYY analogue in 18 ml of 20 mM sodium phosphate buffer (pH7.0), 5 mM sodium caprylate and 750 mM (NH₄)₂SO₄ was performed on acolumn of Butyl sepharose using gradient #1 described above. In FIG. 64the purified conjugate fraction appears in fraction F2.

Example 65 Purification of HSA:Fourth PYY Analogue (SEQ ID NO:47)Conjugate

The fourth PYY analogue is PYY (3-36) Lys³⁷ (ε-MPA)-CONH₂ and has thefollowing sequence:

IKPEAPGEDASPEELNRYYASLRHYLNLVTRQRYK(MPA)-CONH₂

The purification of a conjugate made from reacting 3 ml 25% HSA with 1mM fourth PYY analogue in 18 ml of 20 mM sodium phosphate buffer (pH7.0), 5 mM sodium caprylate and 750 mM (NH₄)₂SO₄ was performed on acolumn of Butyl sepharose using gradient #1 described above. In FIG. 65the purified conjugate fraction appears in fraction F2.

Example 66 Purification of HSA:Fifth PYY Analogue (SEQ ID NO:48)Conjugate

The fifth PYY analogue is MPA-PYY (22-36)-CONH₂ and has the followingsequence: (MPA)-ASLRHYLNLVTRQRY-CONH₂.

The purification of a conjugate made from reacting 6 ml 25% HSA with 1mM fifth PYY analogue in 36 ml of 20 mM sodium phosphate buffer (pH7.0), 5 mM sodium caprylate and 900 mM (NH₄)₂SO₄ was performed on acolumn of Butyl sepharose using gradient #3 described above. In FIG. 66the purified conjugate fraction appears in fraction F2.

Example 67 Purification of HSA:Sixth PYY Analogue (SEQ ID NO:49)Conjugate

The sixth PYY analogue is Acetyl-PYY (22-36) Lys³⁷ (ε-MPA)-CONH₂ and hasthe following sequence: Ac-ASLRHYLNLVTRQRYK(MPA)-CONH₂.

The purification of a conjugate made from reacting 6 ml 25% HSA with 1mM sixth PYY analogue in 36 ml of 20 mM sodium phosphate buffer (pH7.0), 5 mM sodium caprylate and 900 mM (NH₄)₂SO₄ was performed on acolumn of Butyl sepharose using gradient #3 described above. In FIG. 67the purified conjugate fraction appears in fraction F2.

Example 68 Purification of HSA:Second ANP Analogue (SEQ ID NO:50)Conjugate

The second ANP analogue is MPA-ANP (99-126)-CONH₂ and has the followingstructure:

The purification of a conjugate made from reacting 1 ml 25% HSA with 1mM second ANP analogue in 14 ml of 20 mM sodium phosphate buffer (pH7.0), 5 mM sodium caprylate and 750 mM (NH₄)₂SO₄ was performed on acolumn of Butyl sepharose using gradient #3 described above. In FIG. 68the purified conjugate fraction appears in fraction F2.

Example 69 Purification of HSA:Third ANP Analogue (SEQ ID NO:51)Conjugate

The third ANP analogue is ANP (99-126) having reacted with MAL-dPEG₄™(Quanta Biodesign, Powell, Ohio, USA) coupled to Ser⁹⁹. The resultingANP analogue is MPA-EEEEP-ANP (99-126) where EEEEP is ethoxy ethoxyethoxy ethoxy propionic acid; and its sequence is the following:

The purification of a conjugate made from reacting 1 ml 25% HSA with 1mM CJC 1681 in 14 ml of 20 mM sodium phosphate buffer (pH 7.0), 5 mMsodium caprylate and 900 mM (NH₄)₂SO₄ was performed on a column of Butylsepharose using gradient #3 described above. In FIGS. 69A and 69B thepurified conjugate fraction appears in fraction F2.

Example 70 Purification of HSA:First GLP-1 Analogue (SEQ ID NO:1)Conjugate

The first GLP-1 analogue is GLP-1 (7-36) dAla⁸ Lys³⁷ (ε-AEEA-MPA)-CONH₂and his sequence is shown above in Example 1.

The purification of a conjugate made from reacting 1 ml 25% 250 mg/mlHSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM first GLP-1 analoguediluted into 9 ml of buffer made of 20 mM sodium phosphate buffer pH7.0, 5 mM sodium caprylate and 1.75M (NH₄)₂SO₄, was performed on acolumn of Butyl sepharose using the gradient #6 described above. In FIG.70 the purified conjugate fraction appears in fraction B.

Example 71 Purification of HSA:First GLP-1 Analogue (SEQ ID NO:1)Conjugate

The first GLP-1 analogue is GLP-1 (7-36) dAla⁸ Lys³⁷ (ε-AEEA-MPA)-CONH₂and his sequence is shown above in Example 1.

The purification of a conjugate made from reacting 1 ml 25% 250 mg/mlHSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM first GLP-1 analoguediluted into 9 ml of buffer made of 20 mM sodium phosphate buffer pH7.0, 5 mM sodium caprylate and 1.75M magnesium sulfate, was performed ona column of Butyl sepharose using the gradient #6 described above. InFIG. 71 the purified conjugate fraction appears in fraction F2.

Example 72 Purification of HSA:First GLP-1 Analogue (SEQ ID NO:1)Conjugate

The first GLP-1 analogue is GLP-1 (7-36) dAla⁸ Lys³⁷ (ε-AEEA-MPA)-CONH₂and his sequence is shown above in Example 1.

Example with 750 mM ammonium sulfate The purification of a conjugatemade from reacting 1 ml 25% 250 mg/ml HSA (Cortex-Biochem, San Leandro,Calif.) with 1 mM first GLP-1 analogue diluted into 9 ml of buffer madeof 20 mM sodium phosphate buffer pH 7.0, 5 mM sodium caprylate and 750mM (NH₄)₂SO₄, was performed on a column of Butyl sepharose using thegradient #1 described above. In FIG. 72 the purified conjugate fractionappears in fraction F2.

Example 73 Purification of HSA:First GLP-1 Analogue (SEQ ID NO:1)Conjugate

The first GLP-1 analogue is GLP-1 (7-36) dAla⁸ Lys³⁷ (ε-AEEA-MPA)-CONH₂and his sequence is shown above in Example 1.

Example with 1.75M ammonium phosphate The purification of a conjugatemade from reacting 1 ml 25% 250 mg/ml HSA (Cortex-Biochem, San Leandro,Calif.) with 1 mM first GLP-1 analogue diluted into 9 ml of buffer madeof 20 mM sodium phosphate buffer pH 7.0, 5 mM sodium caprylate and 1.75Mammonium phosphate, was performed on a column of Butyl sepharose usingthe gradient #6 described above. In FIG. 73 the purified conjugatefraction appears in fraction B.

Example 74 Purification of HSA:First GLP-1 Analogue (SEQ ID NO:1)Conjugate

The first GLP-1 analogue is GLP-1 (7-36) dAla⁸ Lys³⁷ (ε-AEEA-MPA)-CONH₂and his sequence is shown above in Example 1.

Example with 750 mM ammonium phosphate The purification of a conjugatemade from reacting 1 ml 25% 250 mg/ml HSA (Cortex-Biochem, San Leandro,Calif.) with 1 mM first GLP-1 analogue diluted into 9 ml of buffer madeof 20 mM sodium phosphate buffer pH 7.0, 5 mM sodium caprylate and 750mM ammonium phosphate, was performed on a column of Butyl sepharoseusing the gradient #1 described above. In FIG. 74 the purified conjugatefraction appears in fraction F2.

Example 75 Purification of HSA:First GLP-2 Analogue (SEQ ID NO:52)Conjugate

The first GLP-2 analogue is GLP-2 (1-33) Gly² Lys³⁴ (ε-MPA)-CONH₂ andhas the following sequence:

HGDGSFSDEMNTILDNLAARDFINWLIQTKITDK(MPA)-CONH₂

The purification of a conjugate made from reacting 2 ml 25% 250 mg/mlHSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM first GLP-2 analoguediluted into 14 ml of buffer made of 20 mM sodium phosphate buffer pH7.0, 5 mM sodium caprylate and 750 mM (NH₄)₂SO₄, was performed on acolumn of Butyl sepharose using the gradient #1 described above. In FIG.75 the purified conjugate fraction appears in fraction F2.

Example 76 Purification of RSA:First GLP-2 Analogue (SEQ ID NO:52)Conjugate

The first GLP-2 analogue is GLP-2 (1-33) Gly² Lys³⁴ (ε-MPA)-CONH₂ andhis sequence is shown in Example 75.

The purification of a conjugate made from reacting 9 ml 25% 250 mg/mlRSA (rat serum albumin) with 1 mM first GLP-2 analogue diluted into 14ml of buffer made of 20 mM sodium phosphate buffer pH 7.0, 5 mM sodiumcaprylate and 750 mM (NH₄)₂SO₄, was performed on a column of Butylsepharose using the gradient #1 described above. In FIG. 76 the purifiedconjugate fraction appears in fraction F2.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth, and as follows in the scopeof the appended claims.

1-22. (canceled)
 23. A method for separating albumin conjugate from unconjugated albumin in a solution comprising albumin conjugate and unconjugated albumin, the method comprising: contacting said solution with a hydrophobic interaction chromatography matrix under conditions wherein the albumin conjugate binds to said matrix and the unconjugated albumin does not bind to said matrix, wherein the albumin conjugate comprises a peptide.
 24. The method of claim 23, wherein said hydrophobic interaction chromatography matrix is a column containing a hydrophobic resin.
 25. The method of claim 24, wherein said hydrophobic resin is a bead-formed agarose-based gel filtration matrix covalently coupled to a ligand selected from the group consisting of an octyl group, a phenyl group and a butyl group.
 26. The method of claim 25, wherein said hydrophobic resin is a bead-formed agarose-based gel filtration matrix covalently coupled to a butyl group.
 27. The method of claim 23, wherein the hydrophobic interaction chromatography matrix is, prior to contact with said solution, equilibrated in aqueous buffer at a salt concentration high enough to promote matrix-protein interactions.
 28. The method of claim 27, wherein the said salt concentration is between 500 and 3000 mM.
 29. The method of claim 27, wherein said salt is selected from the group consisting of ammonium phosphate, ammonium sulfate, magnesium phosphate.
 30. The method of claim 27, wherein said salt is ammonium sulfate.
 31. The method of claim 27, wherein the pH of said aqueous buffer is between 3.0 and 9.0.
 32. The method of claim 27, wherein the pH of said aqueous buffer is 7.0.
 33. The method of claim 27, wherein said aqueous buffer and said hydrophobic interaction chromatography matrix are at a temperature of between 4° C. and about 25° C.
 34. The method of claim 27, further comprising applying a gradient of decreasing salt concentration to said hydrophobic interaction chromatography matrix following contact with said solution.
 35. The method of claim 34, further comprising collecting the eluted albumin conjugate.
 36. The method of claim 23, wherein said albumin conjugate consists of a peptide comprising a Michael acceptor covalently bonded to albumin.
 37. The method of claim 36, wherein said bond is between said Michael acceptor and cysteine 34 of said albumin.
 38. The method of claim 37, wherein said Michael acceptor is maleimide-propionic acid.
 39. The method of claim 36, wherein said peptide is covalently bonded to said Michael acceptor, optionally through a linker.
 40. The method of claim 39, wherein said peptide is covalently bonded to said Michael acceptor through a linker selected from the group consisting of (2-amino)ethoxy acetic acid (AEA), ethylenediamine (EDA), 2-[2-(2-amino)ethoxy]ethoxy acetic acid (AEEA), amino ethoxy ethyl amino succinic acid (AEEAS), glycine, 3-aminopropionic acid (APA), 8-aminooctanoic acid (AOA), octanoic acid (OA), and 4-aminobenzoic acid (APhA).
 41. The method of claim 39, wherein said peptide is selected from the group consisting of glucagon like peptide 1 (GLP-1), glucagon like peptide 2 (GLP-2), atrial natriuretic peptide (ANP), kringle 5 (K5), dynorphin, exendin-4, growth hormone releasing factor (GRF), insulin, natriuretic peptides, enfuvirtide (T-20), T-1249, C-34, soluble C-35 peptide EK (SC-35), peptide YY (PYY), and analogs thereof.
 42. The method of claim 39, wherein said peptide is GLP-1 (7-36) dAla⁸ Lys³⁷-CONH₂.
 43. The method of claim 39, wherein said peptide is Exendin-4 (1-39) Lys⁴⁰-CONH₂.
 44. The method of claim 36, wherein said peptide comprising a Michael acceptor is selected from the group consisting of GLP-1 (7-36) dAla⁸ Lys³⁷ (ε-AEEA-MPA)-CONH₂ (SEQ ID NO:1), GRF (1-29) dAla² Gln⁸ Ala¹⁵ Leu²⁷ Lys³⁰ (ε-MPA) CONH₂ (SEQ ID NO:2), Ac-K5 Lys⁸ (ε-MPA)-NH₂ (SEQ ID NO:3), Insulin B1-MPA (SEQ ID NO:4), Insulin A1-MPA (SEQ ID NO:5), MPA-AEEA-C34-CONH₂ (SEQ ID NO:6), C34 (1-34) Lys³⁵ (ε-AEEA-MPA)-CONH₂ (SEQ ID NO:7), C34 (1-34) Lys¹³ (ε-AEEA-MPA)-CONH₂ (SEQ ID NO:8), GLP-1 (7-36) Lys³⁷ (ε-MPA)-NH₂ (SEQ ID NO:9), GLP-1 (7-36) dAla⁸ Lys³⁷ (ε-MPA)-NH₂ (SEQ ID NO:10), GLP-1 (7-36) Lys²⁶ (ε-AEEA-AEEA-MPA) (SEQ ID NO:11), GLP-1 (7-36) Lys³⁴ (ε-AEEA-AEEA-MPA) (SEQ ID NO:12), Exendin-4-(1-39) Lys⁴⁰ (ε-MPA)-NH₂ (SEQ ID NO:13), Exendin-4 (9-39) Lys⁴⁰ (ε-AEEA-MPA)-CONH₂ (SEQ ID NO:14), Dyn A (1-13) (MPA)-NH₂ (SEQ ID NO:15), MPA-AEEA-ANP (99-126)-CONH₂ (SEQ ID NO:16), Dyn A (7-13) Lys¹³ (ε-MPA)-CONH₂ (SEQ ID NO:17), acetyl-Phe-His-cyclohexylstatyl-Ile-Lys (ε-AEEA-MPA)-CONH₂ (SEQ ID NO:18), GLP-1 (7-36) Lys²³ (ε-AEEA-MPA)-CONH₂ (SEQ ID NO:19), GLP-1 (7-36) Lys¹⁸ (ε-AEEA-MPA)-CONH₂ (SEQ ID NO:20), GLP-1 (7-36) Lys²⁶ (ε-AEEA-MPA)-CONH₂ (SEQ ID NO:21), GLP-1 (7-37) Lys⁷ (ε-AEEA-MPA)-CONH₂ (SEQ ID NO:22), GLP-1 (7-36) Lys³⁷ (ε-AEEA-AEEA-MPA)-CONH₂ (SEQ ID NO:23), GLP-1 (7-36) Lys³⁷ (ε-AEEA-MPA)-CONH₂ (SEQ ID NO:24), Exendin-4-(1-39) Lys⁴⁰ (ε-AEEA-MPA)-CONH₂ (SEQ ID NO:25), GLP-1 (7-36) Lys³⁴ (ε-AEEA-MPA)-CONH₂ (SEQ ID NO:26), Insulin B1-OA-MPA (SEQ ID NO:27), Insulin B29-MPA (SEQ ID NO:28), GRF (1-29) Lys³⁰ (ε-MPA)-CONH₂ (SEQ ID NO:29), GRF (1-29) dAla² Gln⁸ dArg¹¹ Ala¹⁵ Leu²⁷ Lys³⁰ (ε-MPA)-CONH₂ (SEQ ID NO:30), GRF (1-29) dAla² Lys³⁰ (ε-MPA)-CONH₂ (SEQ ID NO:31), GLP-1 (9-36) Lys³⁷ (ε-AEEA-MPA)-CONH₂ (SEQ ID NO:32), Ac-T20 (1-36) Lys³⁷ (ε-AEEA-MPA)-CONH₂ (SEQ ID NO:33), Ac-T1249 (1-39) Lys⁴⁰ (ε-AEEA-MPA)-CONH₂ (SEQ ID NO:34), 3′,4′-didehydro-4′-deoxy-C′-norvincaleukoblastine-AEEA-MPA, C34 (1-34) Lys¹³ (ε-MPA)-CONH₂ (SEQ ID NO:36), C34 (1-34) Lys³⁵ (ε-MPA)-CONH₂ (SEQ ID NO:37), MPA-C34 (1-34)-CONH₂ (SEQ ID NO:38), Ac-C34 (1-34) Glu² Lys⁶ Lys⁷ Glu⁹ Glu¹⁰ Lys¹³ Lys¹⁴ Glu¹⁶ Glu¹⁷ Lys²⁰ Lys²¹ Glu²³ Glu²⁴ Lys²⁷ Glu³¹ Lys³⁴ Lys³⁵ Lys³⁶ (ε-AEEA-MPA)-CONH₂ (SEQ ID NO:39), MPA-AEEA-C34 (1-34) Glu² Lys⁶ Lys⁷ Glu⁹ Glu¹⁰ Lys¹³ Lys¹⁴ Glu¹⁶ Glu¹⁷ Lys²⁰ Lys²¹ Glu²³ Glu²⁴ Lys²⁷ Glu³¹ Lys³⁴ Lys³⁵ CONH₂ (SEQ ID NO:40), PYY (3-36) Lys⁴ (ε-OA-MPA)-CONH₂ (SEQ ID NO:41), MPA-OA-PYY (3-36)-CONH₂ (SEQ ID NO:42), Insulin B29-AEES2-MPA (SEQ ID NO:43), Insulin B1-AEES2-MPA (SEQ ID NO:44), Insulin B29-OA-MPA (SEQ ID NO:45), MPA-PYY (3-36)-CONH₂ (SEQ ID NO:46), PYY (3-36) Lys³⁷ (ε-MPA)-CONH₂ (SEQ ID NO:47), MPA-PYY (22-36)-CONH₂ (SEQ ID NO:48), Acetyl-PYY (22-36) Lys³⁷ (ε-MPA)-CONH₂ (SEQ ID NO:49), MPA-ANP (99-126)-CONH₂ (SEQ ID NO:50), MPA-EEEEP-ANP (99-126) (SEQ ID NO:51), and GLP-2 (1-33) Gly² Lys³⁴ (ε-MPA)-CONH₂ (SEQ ID NO:52).
 45. The method of claim 23, wherein said albumin is selected from the group consisting of serum albumin and recombinant albumin.
 46. The method of claim 23, wherein said albumin is human serum albumin.
 47. A hydrophobic interaction chromatography matrix to which an albumin conjugate is bound, wherein said albumin conjugate comprises a peptide. 